EP3473730A1 - Rna-verstärkungsverfahren - Google Patents

Rna-verstärkungsverfahren Download PDF

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EP3473730A1
EP3473730A1 EP18204543.5A EP18204543A EP3473730A1 EP 3473730 A1 EP3473730 A1 EP 3473730A1 EP 18204543 A EP18204543 A EP 18204543A EP 3473730 A1 EP3473730 A1 EP 3473730A1
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rna
primer
cdna
dna
sequence
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EP3473730B1 (de
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Man CHENG
Xiao-Song Gong
Yan Wang
Adam M. Mccoy
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Bio Rad Laboratories Inc
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Bio Rad Laboratories Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • RNAs include but are not limited to miRNA, snoRNA, piRNA, or lncRNA.
  • the methods comprise: obtaining an RNA sample comprising a short RNA target molecule; contacting the sample comprising the short RNA target molecule and at least one RNA-comprising template molecule with a reverse transcriptase, wherein the short RNA target molecule anneals to the RNA-comprising template molecule and the reverse transcriptase reverse transcribes the RNA-comprising template molecule by extending the annealed short RNA molecule to form a duplex comprising a short RNA target molecule/cDNA hybrid polynucleotide annealing to the template RNA molecule; contacting the duplex with RNase H activity, thereby excising ribonucleotides in a portion of the RNA-comprising template molecule in the duplex that anneals to deoxyribonucleotides in the short RNA target molecule/cDNA hybrid polynucleotide; reverse
  • the first oligonucleotide primer is added from an exogenous source.
  • the short RNA is a miRNA, snoRNA, piRNA, or IncRNA.
  • short RNA is 30 or fewer nucleotides in length
  • the RNA-comprising template molecule is a naturally-occurring RNA molecule from the sample.
  • the first oligonucleotide primer has 12-16 nucleotides and comprises a 5' portion not complementary to the single-stranded short RNA target molecule/cDNA hybrid polynucleotide and a 3' portion that is complementary to the single-stranded short RNA target molecule/cDNA hybrid polynucleotide.
  • the first oligonucleotide primer comprises at least 6 nucleotides long that are complementary to the single-stranded short RNA target molecule/cDNA fusion polynucleotide.
  • the 3' portion of the first oligonucleotide primer is 6-10 nucleotides long.
  • the 5' portion of the first oligonucleotide primer is 3-6 nucleotides long.
  • the DNA polymerase has a DNA footprint of 6-10 nucleotides. In some embodiments, the DNA polymerase is linked to a DNA-binding domain having a DNA foot print of 4-6 nucleotides.
  • the RNA-comprising template molecule is heterologous to the sample.
  • the RNA-comprising template molecule comprises (i) a 3' portion complementary to the short RNA target and composed of RNA and (ii) a 5' portion that forms a 5' overhang when the RNA-comprising template molecule is annealed to the short RNA target molecule.
  • the 5' portion is DNA except for a 1-2 ribonucleotide portion of the 5' portion linked to the '3 portion.
  • the first primer is the 5' DNA portion that is released by the RNaseH activity.
  • the 3' portion and 5' portion are RNA.
  • the contacting of the sample comprises contacting the sample with the short RNA target and a plurality of RNA-comprising template molecules having a 5' portion and a 3' portion, wherein said 3' portion comprises a degenerate sequence of at least 4 nucleotides such that the plurality comprises a variety of RNA-comprising molecules having different 3 portions and identical 5 portions.
  • a RNaseH enzyme is added to the duplex, thereby contacting the duplex with RNaseH activity.
  • a RNaseH enzyme is from the reverse transcriptase.
  • the amplifying comprises generating an amplicon
  • the method further comprises nucleotide sequencing the amplicon.
  • the method further comprises quantifying the amount of short RNA target molecule in the sample.
  • the methods comprise: reverse transcribing the RNA by extending a first primer having at least 6 contiguous nucleotides complementary to the RNA, to produce a first strand cDNA comprising a sequence at least 10 nucleotides long, in addition to the primer sequence, that is complementary to the RNA, amplifying the first strand cDNA with a DNA polymerase-DNA binding domain fusion to extend a second primer, wherein the DNA polymerase has a foot print of 6-10 base pairs and the DNA binding domain has a footprint of 3-5 base pairs and wherein the second primer comprises contiguous base pairs complementary to a 3' region of the first strand cDNA to produce a double-stranded cDNA.
  • the first primer comprises a 5' portion not complementary to the RNA.
  • the second primer comprises 12-16 contiguous base pairs complementary to a 3' region of the first strand cDNA
  • the polymerase is a Taq Stoffel fragment or an analog thereof.
  • the DNA binding domain is an Sso7d DNA binding domain.
  • the RNA is a miRNA, snoRNA, piRNA, or lncRNA.
  • the first primer has 6-12 contiguous nucleotides complementary to the RNA.
  • the 5' portion is 3-6 nucleotides long.
  • the amplifying comprises generating an amplicon, and the method further comprises nucleotide sequencing the amplicon. In some embodiments, the method further comprises quantifying the amount of the non-polyA tailed RNA in the sample.
  • the method comprises: adding random poly-W (A/T) or poly-S (G/C) nucleotide sequences to the 3' end of target non-polyA tailed RNA with a terminal transferase to form RNA molecules comprising degenerate nucleotide sequences at the 3' end of the molecules; submitting the RNA molecules comprising the random poly-W or poly-S nucleotide sequences to conditions such that the RNA molecules comprising the random poly-W or poly-S nucleotide sequences anneal to form a duplex of a first and second RNA molecule annealed to each other via the random poly-W or poly-S nucleotide sequences; and extending the 3' ends of the molecules in the duplex with a DNA polymerase to form (i) a first cDNA complementary to the first RNA molecule of the duplex and (ii) a second
  • the RNA is a miRNA, snoRNA, piRNA, or lncRNA.
  • the method further comprises digesting the RNA molecules with an RNAse H activity.
  • the method further comprises amplifying the first or second cDNA with a primer that anneals to the complement of the degenerate sequence.
  • the adding further comprises contacting the non-polyA tailed RNA with a poly A polymerase (e.g. a yeast poly A polymerase).
  • a poly A polymerase e.g. a yeast poly A polymerase
  • the amplifying comprises generating an amplicon
  • the method further comprises nucleotide sequencing the amplicon.
  • the method further comprises quantifying the amount of the non-tailed RNA in the sample.
  • DNA polymerase refers to an enzyme that performs template-directed synthesis of polynucleotides.
  • the term encompasses both a full length polypeptide and a domain that has polymerase activity.
  • DNA polymerases are well-known to those skilled in the art, and include but are not limited to DNA polymerases isolated or derived from Pyrococcus furiosus , Thermococcus litoralis , and Thermotoga maritime , or modified versions thereof. They include both DNA-dependent polymerases and RNA-dependent polymerases such as reverse transcriptase. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. There is little or no sequence similarity among the various families.
  • family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3' to 5' exonuclease activity and 5' to 3' exonuclease activity.
  • Family B polymerases typically have a single catalytic domain with polymerase and 3' to 5' exonuclease activity, as well as accessory factors.
  • Family C polymerases are typically multi-subunit proteins with polymerizing and 3' to 5' exonuclease activity.
  • E. coli three types of DNA polymerases have been found, DNA polymerases I (family A), II (family B), and III (family C).
  • DNA polymerases ⁇ , ⁇ , and ⁇ are implicated in nuclear replication, and a family A polymerase, polymerase ⁇ , is used for mitochondrial DNA replication.
  • DNA polymerases include phage polymerases.
  • RNA polymerases typically include eukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerases as well as phage and viral polymerases. RNA polymerases can be DNA-dependent and RNA-dependent.
  • Thermally stable polymerase refers to any enzyme that catalyzes polynucleotide synthesis by addition of nucleotide units to a nucleotide chain using DNA or RNA as a template and has an optimal activity at a temperature above 45°C.
  • Sso7-like protein refers to polypeptide variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity to SEQ ID NO:4 or 10 of PCT Publication No. WO 2012/177695 .
  • the term includes both full-length Sso7 polypeptides and fragments of the polypeptides that have sequence non-specific double-stranded binding activity.
  • Sso7-like proteins include Sac7d, Sac7e, Ssh7b, and Sto7e. Exemplary mutants of Sso7d include those described in PCT Publication No. WO 2012/138417 .
  • a “domain” refers to a unit of a protein or protein complex, comprising a polypeptide subsequence, a complete polypeptide sequence, or a plurality of polypeptide sequences where that unit has a defined function.
  • the function is understood to be broadly defined and can be ligand binding, catalytic activity, or can have a stabilizing effect on the structure of the protein.
  • DNA binding domain refers to a protein domain that binds DNA in a sequence non-specific manner.
  • the DNA binding domain is a protein domain which binds with significant affinity to DNA, for which there is no known nucleic acid which binds to the protein domain with more than 100-fold more affinity than another nucleic acid with the same nucleotide composition but a different nucleotide sequence.
  • join or “link” refer to any method known in the art for functionally connecting protein domains, including without limitation recombinant fusion with or without intervening domains, intein-mediated fusion, non-covalent association, and covalent bonding, including disulfide bonding; hydrogen bonding; electrostatic bonding; and conformational bonding, e.g ., antibody-antigen, and biotin-avidin associations.
  • nucleic acid amplification refers to any in vitro means for multiplying the copies of a target sequence of nucleic acid.
  • methods include but are not limited to polymerase chain reaction (PCR), DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202 ; PCR Protocols: A Guide to Methods and Applications (Innis et al ., eds, 1990)), (LCR), QBeta RNA replicase, and RNA transcription-based (such as TAS and 3SR) amplification reactions as well as others known to those of skill in the art.
  • PCR polymerase chain reaction
  • DNA ligase chain reaction see U.S. Pat. Nos. 4,683,195 and 4,683,202 ; PCR Protocols: A Guide to Methods and Applications (Innis et al ., eds, 1990)
  • LCR LCR
  • QBeta RNA replicase QBeta RNA replicase
  • amplifying refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide.
  • Components of an amplification reaction include, e.g ., primers, a polynucleotide template, polymerase, nucleotides, and the like.
  • the term amplifying typically refers to an "exponential" increase in target nucleic acid.
  • amplifying as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, such as is obtained with cycle sequencing.
  • PCR Polymerase chain reaction
  • PCR refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression.
  • PCR is well known to those of skill in the art; see, e.g ., U.S. Pat. Nos. 4,683,195 and 4,683,202 ; and PCR Protocols: A Guide to Methods and Applications, Innis et al ., eds, 1990.
  • Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.
  • PCR can be performed as end-point PCR (i.e., only monitored at an end point) or as quantitative PCR (monitored in "real time").
  • an "olignucleotide primer” or “primer” refers to an oligonucleotide sequence that anneals to a sequence on a target nucleic acid and serves as a point of initiation of nucleic acid synthesis.
  • Primers can be of a variety of lengths and are often less than 50 nucleotides in length, for example 12-30 nucleotides in length.
  • the length and sequences of primers for use in PCR can be designed based on principles known to those of skill in the art; see , e.g ., Innis et al ., supra.
  • nucleic acid and “polynucleotide” are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs).
  • PNAs peptide nucleic acids
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g ., hydroxyproline, ⁇ -carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e ., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g ., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups ( e.g ., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • Constantly modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
  • TGG which is ordinarily the only codon for tryptophan
  • amino acid sequences individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same. Sequences are “substantially identical” to each other if they have a specified percentage of nucleotides or amino acid residues that are the same (e.g ., at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) over a specified region where indicated, or across the entire reference sequence if not otherwise indicated, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. These definitions also refer to the complement of a test sequence.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are commonly used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of contiguous positions, for example from 20 to 600 contiguous positions, about 50 to about 200, or about 100 to about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970 ), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970 ), by the search for similarity method of Pearson and Lipman (Proc. Natl. Acad.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993 )).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • RNAs are of great interest in a large number of biological contexts, amplification and detection of short RNAs can be difficult.
  • amplification and detection of short RNAs can be difficult.
  • several methods for amplification and detection of short RNAs from a sample are several methods for amplification and detection of short RNAs from a sample.
  • the methods do not employ an initial primer extension step in which a primer is extended to form a cDNA using the target short RNA as a template. Instead, in some embodiments, the method involves using the target short RNA itself as a primer to perform primer extension on a different template polynucleotide.
  • the template polynucleotide can be a second RNA endogenously present in the sample, or can be an exogenous RNA-containing oligonucleotide (e.g., i.e., from the sample).
  • RNA extension methods allow for primer extension using the short RNA as a template.
  • a polymerase with a small nucleotide footprint fused to a DNA binding-protein with a small nucleotide footprint is used for primer extension.
  • the primer has (1) a 3' portion complementary to the 3' portion of the target short RNA and (2) a 5' portion that forms an overhang (i.e., it is not complementary, and does not anneal, to the target short RNA).
  • Use of the small footprint polymerase with the DNA binding domain allows for extension of the target short RNA in circumstances where a polymerase such as Taq polymerase would be inefficient or would not be able to extend the primer.
  • the resulting cDNA will be longer than the target short RNA (e.g., by the number of nucleotides in the 5' overhang of the primer).
  • the resulting cDNA can then be amplified using a "forward" primer that anneals (e.g., is complementary) to the '3 portion of the cDNA in combination with the primer with the 5' overhang, which functions as a "reverse" primer.
  • Exemplary target RNAs that can be detected and amplified using the methods described herein include, but are not limited to, miRNA, snRNA, snoRNA, piRNA, or lncRNA.
  • MicroRNAs typically 18 to 25 nt in length, are non-protein-coding RNAs that can inhibit the translation of target mRNAs (see, e.g., Croce and Calin, Cell 122(1): 6-7 (2005 )).
  • Other small RNAs include small nucleoplasmic RNAs (snRNAs) and small nucleolar RNAs (snoRNAs).
  • RNA molecules can function, for example, in mRNA splicing (U1, U2, and U4 to U6 snRNAs), mRNA and rRNA processing (U7 snRNA; U3 and U8 snoRNAs), and site selection for RNA modification by methylation of the 2' hydroxyl group (box C/D snoRNAs) or by pseudouridine formation (box H/ACA snoRNAs).
  • Piwi-interacting RNAs piRNAs
  • piRNAs were identified through association with Piwi proteins in mammalian. piRNAs can range from 26-30 nucleotides in length. Long noncoding RNA (lncRNA) have also been described.
  • the methods do not require or involve reverse transcription of the target RNA until after the target RNA has itself acted as a primer.
  • another RNA-containing polynucleotide can be used as a template for reverse transcription, with the target RNA functioning as a primer.
  • the target RNA will be linked to the resulting cDNA.
  • the resulting polynucleotide will therefore be partially composed of RNA nucleotides (i.e., the target RNA sequence) and partially composed of DNA (i.e., the nucleotides added in the reverse transcription).
  • FIG. 1 exemplifies an embodiment as described above.
  • a target miRNA is exposed to conditions to anneal the target miRNA to an RNA-containing template forming a target/template duplex.
  • the RNA-containing template can be, for example, an mRNA or other longer RNA from the same or a different biological sample from which the target RNA (e.g., miRNA) was obtained.
  • Other different RNA-containing template options are described below.
  • the annealed target/template complex can be exposed to a protein having reverse transcriptase activity such that the reverse transcriptase activity extends the 3' end of the target RNA to generate a cDNA complementary to at least a portion of the template RNA.
  • a protein having reverse transcriptase activity such that the reverse transcriptase activity extends the 3' end of the target RNA to generate a cDNA complementary to at least a portion of the template RNA.
  • Any conditions for reverse transcription reactions can be used.
  • the resulting cDNA will be composed of a 5' portion made of the target RNA, and a 3' portion made up of the cDNA.
  • the resulting target RNA/cDNA hybrid polynucleotide can be of any length that incorporates the target RNA length and at least some deoxynucleotides complementary to the template RNA.
  • the length of the cDNA portion of the RNA target/cDNA hybrid polynucleotide will depend on the length of the template RNA as well as the position at which the target RNA anneals to the template RNA.
  • the cDNA will be at least 10, 20, 30, 40, 50, 100, or 150 nucleotides, e.g., 10-100 or 50-500 nucleotides.
  • reverse transcriptases include but are not limited to murine leukemia virus (MLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Respiratory Syncytial Virus (RSV) reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reverse transcriptase, Rous-associated Virus-2 (RAV2) reverse transcriptase, SUPERSCRIPT II reverse transcriptase, SUPERSCRIPT I reverse transcriptase, THERMOSCRIPT reverse transcriptase and MMLV RNase H - reverse transcriptases.
  • MMV murine leukemia virus
  • AMV Avian Myeloblastosis Virus
  • RSV Respiratory Syncytial Virus
  • EIAV Equine Infectious Anemia Virus
  • RAV2 Rous-associated Virus-2
  • SUPERSCRIPT II reverse transcriptase SUPERSCRIPT I reverse transcriptase
  • THERMOSCRIPT reverse transcriptase MMLV RNase
  • Tth and Z05 which are DNA polymerases, can function as reverse transcriptase in the presence of manganese.
  • concentration of the reverse transcriptase can vary and optimal concentrations can be determined empirically and depend on the particular reverse transcriptase used.
  • the portion the template RNA complementary to the newly-formed cDNA can subsequently be cleaved from the remainder of the RNA-containing template polynucleotide.
  • RNase H is used for this purpose.
  • RNase H is an endoribonuclease that specifically hydrolyzes the phosphodiester bonds of RNA which is annealed to DNA. RNase H does not digest single or double-stranded DNA.
  • RNAse H can be applied to the RNA template/cDNA double-stranded complex.
  • RNA nucleotides annealing to deoxynucleotides of the cDNA will be removed from the RNA template.
  • the reverse transcriptase used will have inherent RNaseH activity that will cleave and degrade the RNA as described above. This aspect is exemplified, for example, in steps 13, 23, and 33 of FIGs. 1-3 , respectively.
  • a primer (referred to for convenience as "first primer") complementary to the cDNA portion of the target RNA/cDNA hybrid can be annealed to the cDNA and a second reverse transcription reaction can be performed.
  • a first primer that anneals to the cDNA is extended to form a second cDNA complementary to the target RNA.
  • the first primer can anneal to the cDNA sequence or can anneal to both the target RNA and the cDNA, annealing to the junction between the two sequences.
  • the first primer can be added exogenously, or as discussed below, can be generated in some circumstances by the RNaseH activity.
  • the resulting second cDNA therefore comprises a sequence complementary to the entire, or substantially the entire, target RNA and further comprises the first primer sequence.
  • the second cDNA can subsequently be amplified as desired using standard techniques. This is exemplified in steps 15, 25, and 35 of FIGs 1-3 , respectively.
  • the polymerase chain reaction is used to amplify the second cDNA.
  • a "reverse" primer complementary to the 3' portion of the second cDNA can be used in combination with the first primer and a thermostable polymerase under thermocyclic conditions to generate a double stranded DNA comprising the target RNA sequence. If desired, the PCR can be performed quantitatively and be monitored in real-time.
  • the method can employ an RNA-containing template polynucleotide.
  • the embodiment illustrated in FIG.1 is a naturally-occurring RNA from a biological sample.
  • the RNA-containing template polynucleotide can be a synthetic polynucleotide.
  • the RNA-containing template is a synthetic RNA oligonucleotide.
  • the synthetic RNA-containing template is partially DNA and partially RNA (exemplified in FIG. 3 ).
  • the 5' end portion of the template is DNA and the 3' end portion of the template is RNA.
  • the RNaseH activity in this latter option releases the DNA portion of the template, which can then be available to function as the first primer in the second reverse transcription step, without addition of an exogenous first primer.
  • the 3' end portion of the template will be complementary to the target RNA, or at least substantially complementary such that the 3' end portion of the template anneals to the target RNA under conditions in which the reverse transcriptase is active and can extend the target RNA using the RNA-containing template polynucleotide as a template.
  • the 5' portion is not complementary to the target RNA and acts as a non-annealing tail.
  • the 3' end portion of the template will be of sufficient length to allow annealing to the target RNA and can be, for example, at least 4, 5, 6, 7, 8, 9, 10, or more nucleotides long, e.g., 5-30 or more nucleotides. Of course, the annealing sequence of the template will be no longer than the target RNA itself.
  • the portion of the template composed of RNA will extend at least one (e.g., 1, 2, 3, or more) nucleotide beyond the annealing portion of the template (said another way, the 5' non-annealing portion of the template is DNA except for a short (e.g., 1, 2, 3, or more nucleotides) that is composed of ribonucleotides).
  • a short e.g., 1, 2, 3, or more nucleotides
  • FIG. 3 bounded by vertical lines exemplified in FIG. 3 bounded by vertical lines.
  • the effect of these particular nucleotides being RNA is that the complementary nucleotides in the resulting cDNA formed in the reverse transcription step will be DNA, thereby forming for these particular nucleotides an RNA/DNA duplex, which enables cleavage by the RNase H activity.
  • RNA/DNA duplex that can be degraded by RNase H.
  • RNase H excises RNA in RNA/DNA duplexes, and thus the presence of one or more RNA nucleotides just after the annealing sequence of the template will allow for a region of the RNA/DNA duplex that will subsequently be cleaved by RNase H activity. Degradation of at least a portion of, and ideally all of, the template allows for the first primer to subsequently anneal to the cDNA without (or with reduced) competition from the portion of the template complementary to the cDNA.
  • the 5' end portion of the template can vary in length but in some embodiments in which the template is composed of RNA and DNA, at least the first three nucleotides in the 5' end of the template will be DNA. In some embodiments, the 5' end portion is 4, 5, 6, 7, 8, or more DNA nucleotides, e.g., from 3-100 or more nucleotides.
  • the synthetic RNA-containing template polynucleotide can be provided in the form of a plurality of RNA-containing template polynucleotides wherein some or all of the RNA sequence (i.e., at least the 3' end of the template) is a degenerate sequence.
  • some or all of the RNA sequence i.e., at least the 3' end of the template
  • a degenerate sequence By providing a random sequence at the 3' end of the template, more than one short RNA target can be amplified.
  • a library of small RNAs can be generated.
  • the method involves a second reverse transcription step to generate a second cDNA based on a primer ("first primer") complementary to the cDNA portion of the target RNA/cDNA hybrid polynucleotide followed by amplification using the first primer and a reverse primer.
  • the second reverse transcription step is exemplified in steps 14, 24, and 34 of FIGs. 1-3 , respectively.
  • the first primer is at least partially complementary to the cDNA portion of the target RNA/cDNA hybrid polynucleotide such that the first primer anneals to the complementary sequence in the cDNA.
  • the sequence of the cDNA portion is not necessarily known.
  • At least the 3' portion of the first primer can be designed to be complementary to a portion of the 3' end of the target RNA such that the second reverse transcription step results in extension of the first primer using the target RNA as a template.
  • This aspect is exemplified in FIG. 4 , option "A.”
  • a 5' portion of the first primer can be complementary to the cDNA and a 3' portion of the first primer can be complementary to the target RNA, thereby annealing to the junction of the cDNA and the target RNA.
  • the first primer can be complementary only to the cDNA portion.
  • the amplification step can involve extension of the first primer and the reverse primer to form a double-stranded amplicon comprising the target RNA sequence (albeit in DNA rather than RNA).
  • the amplification will typically involve primer extension with a DNA polymerase (including but not limited to the polymerase chain reaction (PCR)).
  • DNA polymerases useful in the present invention can be any polymerase capable of replicating a DNA molecule.
  • Exemplary DNA polymerases are thermostable polymerases, which are especially useful in PCR.
  • Thermostable polymerases are isolated from a wide variety of thermophilic bacteria, such as Thermus aquaticus ( Taq ), Thermus brockianus ( Tbr ), Thermus flavus ( Tfl ), Thermus ruber ( Tru ), Thermus thermophilus ( Tth ), Thermococcus litoralis ( Tli ) and other species of the Thermococcus genus, Thermoplasma acidophilum ( Tac ), Thermotoga neapolitana ( Tne ), Thermotoga maritima ( Tma ), and other species of the Thermotoga genus, Pyrococcus Juriosus ( Pfu ), Pyrococcus woesei ( Pwo ) and other species of the Pyrococcus genus, Bacillus sterothermophilus ( Bst ), Sulfolobus acidocaldarius ( Sac ) Sulfolobus solfataricus
  • the polymerase enzyme is a hybrid polymerase comprising a polymerase domain and a DNA binding domain.
  • hybrid polymerases are known to show an increased processivity. See e.g., U.S. Patent Application Publication Nos. 2006/005174 ; 2004/0219558 ; 2004/0214194 ; 2004/0191825 ; 2004/0081963 ; 2004/0002076 ; 2003/0162173 ; 2003/0148330 ; 2003/0138830 and U.S. Patent Nos.
  • hybrid polymerases lack 3'-5' exonuclease activity.
  • hybrid polymerases comprise a double point mutation in the polymerase domain that provides this exonuclease deficiency.
  • hybrid polymerases can comprise the double point mutation D141A/E143A in the polymerase domain.
  • the binding domain of hybrid polymerases is from a thermostable organism and provides enhanced primer annealing at higher temperatures, e.g., temperatures above 45°C.
  • Sso7d and Sac7d are small (about 7 kd MW), basic chromosomal proteins from the hyperthermophilic archaeabacteria Sulfolobus solfataricus and S. acidocaldarius , respectively ( see, e.g., Choli et al., Biochimica et Biophysica Acta 950:193-203, 1988 ; Baumann et al., Structural Biol. 1:808-819, 1994 ; and Gao et al, Nature Struc. Biol.
  • Sso7d, Sac7d, Sac7e and related sequences are known in the art (see, e.g., accession numbers (P39476 (Sso7d); P13123 (Sac7d); and P13125 (Sac7e)). These sequences typically have at least 75% or greater, of 80%, 85%, 90%, or 95% or greater, amino acid sequence identity. For example, an Sso7 protein typically has at least 75% identity to an Sso7d sequence.
  • hybrid polymerases of use are described for example in U.S. Patent Application Publication Nos. 2006/005174 ; 2004/0219558 ; 2004/0214194 ; 2004/0191825 ; 2004/0081963 ; 2004/0002076 ; 2003/0162173 ; 2003/0148330 ; 2003/0138830 ; PCT Publication No. WO 2012/138417 ; and U.S. Patent Nos. 6,627,424 and 7,445,898 , each of which is hereby incorporated by reference in its entirety for all purposes and in particular for all teachings related to polymerases, hybrid/chimeric polymerases, as well as all methods for making and using such polymerases.
  • Examples of hybrid polymerase proteins and methods of generating hybrid proteins are also disclosed in WO2004011605 , which is hereby incorporated by reference in its entirety for all purposes, and in particular for all teachings related to generating hybrid proteins.
  • the DNA polymerase has a relatively small nucleotide footprint, and as a consequence, low binding affinity for a DNA substrate.
  • such polymerase is fused to a DNA binding protein also having a small footprint, wherein the fusion has an increased DNA affinity compared to the DNA polymerase alone.
  • the DNA polymerase has a nucleotide footprint of 6-10 nucleotides and is linked to a sequence non-specific DNA binding protein having a nucleotide footprint of 3-6. The resulting fusion protein will have a small nucleotide footprint (e.g.
  • the template has fewer than 40, 35, 30, 28, 26, 25, or 24 nucleotides.
  • An exemplary DNA polymerase having a small nucleotide footprint is the Stoffel fragment of Taq polymerase or an analogous fragment from another thermostable (e.g., A family) polymerase.
  • the Stoffel fragment is a Taq polymerase fragment lacking the amino-terminal 289 amino acids of Taq, and lacks 5'-3' exonuclease activity.
  • a Stoffel fragment analog refers to a family A polymerase lacking an amino-terminal 5'-3' exonuclease domain.
  • the analog has amino acids corresponding to those of the Stoffel fragmentand lacks the N-terminal amino acids corresponding to the first 289 amino acids of Taq.
  • KlenTaq lacks the first 280 amino acids of Taq.
  • original amount of a target RNA in a sample is determined following the amplification methods described herein.
  • quantitative amplification can be used to determine the original amount of a target RNA in a sample.
  • Quantitative amplification methods e.g., quantitative PCR or quantitative linear amplification
  • an nucleic acid template e.g., a step corresponding to steps 15, 25, or 35 of FIGs. 1-3 , respectively
  • directly or indirectly e.g., determining a Ct value
  • PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS (Innis et al., eds, 1990)).
  • PCR is used to amplify DNA templates.
  • alternative methods of amplification have been described and can also be employed, as long as the alternative methods amplify intact DNA to a greater extent than the methods amplify cleaved DNA. Methods of quantitative amplification are disclosed in, e.g., U.S. Pat. Nos.
  • quantitative amplification is based on the monitoring of the signal (e.g., fluorescence of a probe) representing copies of the template in cycles of an amplification (e.g., PCR) reaction.
  • the signal e.g., fluorescence of a probe
  • an amplification e.g., PCR
  • a very low signal is observed because the quantity of the amplicon formed does not support a measurable signal output from the assay.
  • the signal intensity increases to a measurable level and reaches a plateau in later cycles when the PCR enters into a non-logarithmic phase.
  • the specific cycle at which a measurable signal is obtained from the PCR reaction can be deduced and used to back-calculate the quantity of the target before the start of the PCR.
  • the number of the specific cycles that is determined by this method is typically referred to as the cycle threshold (Ct).
  • Ct cycle threshold
  • Exemplary methods are described in, e.g., Heid et al. Genome Methods 6:986-94 (1996 ) with reference to hydrolysis probes.
  • the fluorogenic probe consists of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5'-exonuclease activity of DNA polymerase if, and only if, it anneals to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye.
  • Another method of detecting amplification products that relies on the use of energy transfer is the "beacon probe” method described by Tyagi and Kramer, Nature Biotech. 14:303-309 (1996 ), which is also the subject of U.S. Pat. Nos. 5,119,801 and 5,312,728 .
  • This method employs oligonucleotide hybridization probes that can form hairpin structures. On one end of the hybridization probe (either the 5' or 3' end), there is a donor fluorophore, and on the other end, an acceptor moiety. In the case of the Tyagi and Kramer method, this acceptor moiety is a quencher, that is, the acceptor absorbs energy released by the donor, but then does not itself fluoresce.
  • the beacon when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched.
  • the molecular beacon probe which anneals to one of the strands of the PCR product, is in the open conformation and fluorescence is detected, while those that remain unhybridized will not fluoresce ( Tyagi and Kramer, Nature Biotechnol. 14: 303-306 (1996 )).
  • the amount of fluorescence will increase as the amount of PCR product increases, and thus may be used as a measure of the progress of the PCR.
  • Those of skill in the art will recognize that other methods of quantitative amplification are also available.
  • some methodologies employ one or more probe oligonucleotides that are structured such that a change in fluorescence is generated when the oligonucleotide(s) is annealed to a target nucleic acid.
  • one such method involves is a dual fluorophore approach that exploits fluorescence resonance energy transfer (FRET), e.g., LightCyclerTM hybridization probes, where two oligo probes anneal to the amplicon.
  • FRET fluorescence resonance energy transfer
  • the oligonucleotides are designed to hybridize in a head-to-tail orientation with the fluorophores separated at a distance that is compatible with efficient energy transfer.
  • ScorpionsTM probes e.g., Whitcombe et al., Nature Biotechnology 17:804-807, 1999 , and U.S. Pat. No. 6,326,145
  • SunriseTM or AmplifluorTM
  • probes that form
  • intercalating agents that produce a signal when intercalated in double stranded DNA may be used.
  • exemplary agents include SYBR GREENTM, SYBR GOLDTM, and EVAGREENTM. Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than, for example, primer-dimers, etc.
  • the quantity of a DNA region is determined by nucleotide sequencing copies in a sample and then determining the relative or absolute number of copies having the same sequence in a sample.
  • sequencing of enriched fragments can be particularly effective when high throughput sequencing is used, e.g., "next generation sequencing methods such as HiSeqTM, MiSeqTM, or Genome Analyzer (each available from Illumina), SOLiDTM or Ion TorrentTM (each available from Life Technologies) and 454TM sequencing (from Roche Diagnostics).
  • sequencing involves single-molecule, real-time (SMRT) sequencing.
  • SMRT sequencing is a process by which single DNA polymerase molecules are observed in real time while they catalyze the incorporation of fluorescently labeled nucleotides complementary to a template nucleic acid strand.
  • Methods of SMRT sequencing are known in the art and were initially described by Flusberg et al., Nature Methods, 7:461-465 (2010 ), which is incorporated herein by reference for all purposes.
  • the nucleotide sequence of an amplicon based on target RNA or DNA is determined.
  • Methods of nucleic acid sequencing are well-known in the art. Examples of sequence analysis include, but are not limited to, Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing ( Sears et al., Biotechniques, 13:626-633 (1992 )), solid-phase sequencing ( Zimmerman et al., Methods Mol.
  • the amplicon is sequenced by single-molecule, real-time (SMRT) sequencing.
  • SMRT single-molecule, real-time
  • SMRT sequencing is a process by which single DNA polymerase molecules are observed in real time while they catalyze the incorporation of fluorescently labeled nucleotides complementary to a template nucleic acid strand.
  • Methods of SMRT sequencing are known in the art and were initially described by Flusberg et al., Nature Methods, 7:461-465 (2010 ), which is incorporated herein by reference for all purposes.
  • nucleotide sequencing does not comprise template-dependent replication of a DNA region.
  • the amplicon is sequenced by nanopore sequencing.
  • Nanopore sequencing is a process by which a polynucleotide or nucleic acid fragment is passed through a pore (such as a protein pore) under an applied potential while recording modulations of the ionic current passing through the pore. Methods of nanopore sequencing are known in the art; see , e.g ., Clarke et al., Nature Nanotechnology 4:265-270 (2009 ), which is incorporated herein by reference for all purposes.
  • the amplicon is hybridized to another nucleic acid.
  • the nucleic acid is linked to a solid support.
  • the DNA is hybridized to a microarray.
  • a microarray is useful, for example, in monitoring the presence, absensce or quantity of multiple sequences in the DNA.
  • DNA from different samples can be hybridized to one or more nucleic acids, thereby determining the differential amount of one or more particular sequence between the samples, Thus, for example, diseased and healthy cells, or cells obtained at different times, or before and after or during treatment can be compared.
  • RNA detection methods involves using two short target RNAs to act as reverse transcription primers for each other.
  • This aspect is exemplified in FIG. 6 .
  • a mixture comprising at least two RNAs of different sequence is provided.
  • the mixture can have more than two RNAs of different sequence.
  • the RNAs likely are unrelated in sequence to each other, they will not necessarily anneal to each other in unmodified form. Accordingly, the RNAs in the mixture can be submitted to terminal transferase (TdT) in the presence of nucleotides A/T only or G/C only (template independent nucleotide polymerization).
  • TdT terminal transferase
  • RNA molecules will have poly-W (A/T) or poly-S (G/C) 3' tails.
  • TdT has lower efficiency to add DNA to the 3' end of RNA molecules and thus, in some embodiments, yeast poly-A polymerase can be used to add several (e.g., ⁇ 3 nt) DNA-A sequence to the 3' end of RNA molecule to create an RNA/DNA hybrid with a very short 3' end DNA tail that can act as a substrate for TdT. Due to the random nature and low complexity of the tails, the tails will allow for annealing of some of the 3' tails to other 3' tails, allowing for pairs of RNAs to prime each other.
  • a polyA polymerase which catalyzes the template independent addition of AMP from ATP to the 3' end of RNA, see , e.g ., Martin and Keller, RNA 4:226-230 (1998 )
  • a yeast poly A polymerase e.g., a yeast poly A polymerase
  • the temperature of the reaction is initially set such that the poly A polymerase is active and then subsequently the temperature is changed so that the terminal tarnsferase is active.
  • the inclusion of poly A polymerase can provide a few A nucleotides to the RNA, making for a better substrate for terminal transferase.
  • Removal of target RNA sequence can be achieved by inclusion of RNase H or by intrinsic RNase H activity of reverse transcriptase to generate a single-stranded complementary cDNA sequence with a 5' poly-W or G tail.
  • the cDNAs can subsequently be detected as described above, e.g., by PCR, qPCR, or hybridization-based methods like microarray, or sequencing-based methods, including but not limited to RNAseq or next-generation sequencing methods as described above.
  • a short target RNA can be amplified by an initial reverse transcription step using a short, optionally tailed, primer.
  • the short primer has at least a 3' portion that is complementary to the target RNA 3' end and, optionally also a 5' portion that is not complementary to the target RNA and instead will function as a length extender.
  • This aspect is exemplified in FIG. 5 .
  • Reverse transcription with the tailed primer results in a cDNA comprising additional nucleotides complementary to the "tail" of the tailed primer.
  • the tailed primer and a forward primer that anneals to a sequence (ideally at or near the 3' end of) of the cDNA can subsequently be used to amplify the cDNA.
  • RNA having 25 or fewer nucleotides for example RNA having 25 or fewer nucleotides, e.g., 20-25 nucleotides long.
  • RT-PCR is a powerful tool in monitoring mRNA expression level, however, this approach cannot be directly or easily implemented for miRNA and other very short (25nt or fewer nucleotides) RNA due to its extreme short length (miRNA are typically in the range of 21-25 nt).
  • Typical PCR primers need to be > 17 nt to function effectively in PCR reaction that is supported by Taq polymerase, and two primers are required to support exponential amplification. Therefore the length of miRNA (or the corresponding cDNA) is too short to accommodate two standard primers.
  • a polymerase having a small nucleotide footprint e.g., 6-10 nucleotides long
  • a DNA binding domain having a small nucleotide footprint 4-6 nucleotides
  • primers as short as 12 or even less (e.g., 10, 11) nucleotides long.
  • an exemplary polymerase having a small nucleotide footprint is the Stoffel fragment of Taq polymerase or an analogous fragment from another thermostable (e.g., A family) polymerase and an exemplary DNA binding domain is Sso7d domain.
  • the primer described above can have, e.g., 6, 7, 8, 9, 10, 11, 12, e.g., 6-12, 6-10, 9-12 contiguous nucleotides complementary to the target RNA and optionally an additional "tail" that is not complementary to the target RNA.
  • the tail is 4, 5, or 6 (e.g., 4-6) nucleotides, though the tail can be longer or shorter as well.
  • the forward primer can also be designed to have 8, 10, 11, 12, 13, 14, 15 or 16 (e.g., 8-15 or 12-16) or more nucleotides, thereby allowing for the forward and tailed primers to reside on the cDNA or complementary sequence to allow for efficient amplification.
  • the tailed primer and forward primer will overlap by 1, 2, 3, or more base pairs. See part B of FIG. 5 .
  • the tailed primer and forward primer will overlap by 1, 2, 3, or more base pairs. See part B of FIG. 5 .
  • the tailed primer and forward primer will overlap by 1, 2, 3, or more base pairs. See
  • a small nucleotide footprint polymerase linked to a small nucleotide footprint DNA binding protein can be further used in an analogous fashion to amplify any short polynucleotides.
  • formalin or formaldehyde-fixed, paraffin-embedded (FFPE) tissue can contain significantly fragmented polynucleotides (RNA or DNA) that can be a challenged to amplify using standard PCR reagents such as Taq polymerase.
  • RNA or DNA polynucleotides
  • standard PCR reagents such as Taq polymerase.
  • the method described above for amplifying short RNAs can be similarly used for amplifying short (e.g., 20-25 nucleotides) DNA sequences, including but not limited to those in FFPE samples.
  • Samples can be any mixture containing a short RNA.
  • the sample is derived from a biological fluid, cell or tissue.
  • the sample can be crude or purified.
  • the sample is a preparation of RNA from a cell or cells.
  • the cells are animal cells, including but not limited to, human, or non-human, mammalian cells.
  • Non-human mammalian cells include but are not limited to, primate cells, mouse cells, rat cells, porcine cells, and bovine cells.
  • the cells are plant or fungal (including but not limited to yeast) cells.
  • Cells can be, for example, cultured primary cells, immortalized culture cells or can be from a biopsy or tissue sample, optionally cultured and stimulated to divide before assayed. Cultured cells can be in suspension or adherent prior to and/or during the permeabilization and/or DNA modification steps. Cells can be from animal tissues, biopsies, etc. For example, the cells can be from a tumor biopsy.
  • samples include RNA or DNA targets that only have short amplifiable regions (e.g., wherein a target region has less than 50, 40, 30, 25, or 20 contiguous amplifiable nucleotides), degraded, or are otherwise difficult to amplify due to nucleic acid degradation.
  • formalin-fixed samples can have only short sequences of nucleic acid due to fixation.
  • ancient nucleic acid samples or samples that have been exposed to chemical or temperature conditions that degrade nucleic acids can be amplified by the methods described herein in view of the method's ability to amplify shorter sequences than typically can be amplified in PCR.
  • Example 1 Model system for generating miRNA-specific cDNA through miRNA-based priming.
  • RNA1 5' GCAUCAGCGACACACUCAAGAG
  • RNA2 38 nt long,5' UGAUGACCCCAGGUAACUCUUGAGUGUGUCGCUGAUGC
  • RNA1 20pM
  • reverse transcription reagents iScript cDNA synthesis kit, SuperScript III, and SuperScript III preblended with RNaseH
  • RNA1 RNA1 in this example
  • RNA1 can serve as a primer for the reverse transcription reaction to extend off a long RNA template
  • RNA2 in this example to create an RNA-DNA hybrid
  • the RNaseH activity of the Reverse Transcriptase cleaves or nicks the RNA (as part of RNA2) involved in RNA-DNA base pairing, which generate additional short RNA primers that can be extended to create cDNA of the original short RNA strand (RNA1)
  • this cDNA can then be amplified by regular qPCR to allow quantitation of the original input quantity of the short RNA.
  • Synthetic miR-16 (200 pM) was mixed with (100 ng) purified Hela RNA that contains low amount of low molecular weight RNAs (e.g. miRNA, snoRNA, etc), which was then subjected to reverse transcription using the MMLV RTase in the iScript cDNA synthesis kit, (by Bio-Rad Inc.) and a buffer (a buffer comprising 10% glycerol and 900 ⁇ M each of dATP and dTTP, 450 ⁇ M each of dGTP and dCTP). As controls, 100 ng of the above Hela RNA and the synthetic miR-16 was also separately (without mixing together) subjected to reverse transcription using the same reagents.
  • RNAs e.g. miRNA, snoRNA, etc
  • Example 3 miRNA amplification via semi-degenerate priming
  • Synthetic miR-16 RNA (2-200pM) was mixed with an enzyme cocktail containing terminal deoxytransferase, yeast polyA polymerase and MMLV reverse transcriptase in the presence of a modified buffer (a buffer comprising 10% glycerol and 900 ⁇ M each of dATP and dTTP, 450 ⁇ M each of dGTP and dCTP). The mixture was then subjected to incubation at 37C for 20min (allowing yeast poly A polymerase to function), 25C for 15 min (allowing TdT enzyme to function), 37C for 20 min (allowing RT enzyme to function), and 85C for 5 min (inactivating all enzymes).
  • a modified buffer a buffer comprising 10% glycerol and 900 ⁇ M each of dATP and dTTP, 450 ⁇ M each of dGTP and dCTP.
  • the products were cloned into a TOPO vector, and 11 clones were sequenced. miR-16 sequence aligned with high identity with sequence contained in each of the 11 clones.
  • the results indicate that when the 3' terminus of miR-16 was extended to contain semi-degenerate sequences using combination of TdT and yeast polyA polymerase, the semi-degenerate region from different extended molecules could anneal to each other and serve as primers in the reverse transcription reaction, thus allowing the cDNA for the miR-16 to be generated and subsequently amplified in qPCR.
  • Example 4 qPCR Amplification of miRNA cDNA using short primers and Sso-fusion polymerase
  • Synthetic miR-16 cDNA (22 nt, 5'TAGCAGCACGTAAATATTGGCG, at 20pM) was used as the starting template. It was mixed with 250 nM each of two miR16-specific PCR primers, 16Cfor (11 nt, 5' TAGCAGCACGT) and 16Crevc (12 nt, 5' CGCCAATATTTA), and DyNAmo qPCR supermix, which contained a fusion polymerase of Sso7d domain fused to the polymerase domain of Tbr polymerase. The mixture was subjected to the following amplification protocol: 95C-15min, 94C-10s, 45x(41C-20sec, 60C-10s).
  • the intended target was successfully amplified, resulting in Cq value of 21 and Tm of 62C for the amplified product.
  • This example demonstrates that very short primers (e.g. 11-12 nt long) can be efficiently utilized by Sso-fusion polymerase to support PCR amplification.
  • Example 5 Short primers can be utilized Sso-fusion polymerase more efficiently in PCR
  • Lambda DNA (2.5 ng per reaction) was used as the template together with PCR primers (150 nM each) in the presence of either wild type Taq polymerase (Taq, 1 unit per reaction) or Sso7d fusion to the Stoffel fragment (Sst, 1 unit per reaction).
  • the primers used were 732R16 (16 nt, 5'TTCGATATATTCACTC) and 57F12 (12 nt, 5' TTCGTCATAACT).
  • the final reaction buffer contained 20 mM Tris (pH 8.4), 50 mM KCl, 2 mM MgCl2, and 0.2 mM each dNTPs.
  • the annealing temperature was varied from 46C to 64C using gradient and followed by extension at 72C.
  • the amount of amplified products was determined by adding dsDNA binding dye to the reaction after the amplification was complete and analyzed on a fluorescent plate reader.
  • the intensity of the fluorescent signal correlated to the quantity of the amplified product and the efficiency of the PCR amplification.
  • the annealing temperature used was between 46C and 50C, the amplification product produced by the Sst fusion polymerase resulted in a fluorescence signal of 9000 units, whereas that by the wild type Taq polymerase was below 1000 units.
  • a method of amplifying a short RNA molecule comprising, obtaining an RNA sample comprising a short RNA target molecule; contacting the sample comprising the short RNA target molecule and at least one RNA-comprising template molecule with a reverse transcriptase, wherein the short RNA target molecule anneals to the RNA-comprising template molecule and the reverse transcriptase reverse transcribes the RNA-comprising template molecule by extending the annealed short RNA molecule to form a duplex comprising a short RNA target molecule/cDNA hybrid polynucleotide annealing to the template RNA molecule; contacting the duplex with RNase H activity, thereby excising ribonucleotides in a portion of the RNA-comprising template molecule in the duplex that anneals to deoxyribonucleotides in the short RNA target molecule/cDNA hybrid polynucleotide; reverse transcribing at least
  • the short RNA is a miRNA, snoRNA, piRNA, or lncRNA.
  • short RNA is 30 or fewer nucleotides in length
  • RNA-comprising template molecule is a naturally-occurring RNA molecule from the sample.
  • the first oligonucleotide primer has 12-16 nucleotides and comprises a 5' portion not complementary to the single-stranded short RNA target molecule/cDNA hybrid polynucleotide and a 3' portion that is complementary to the single-stranded short RNA target molecule/cDNA hybrid polynucleotide.
  • the first oligonucleotide primer comprises at least 6 nucleotides long that are complementary to the single-stranded short RNA target molecule/cDNA fusion polynucleotide.
  • RNA-comprising template molecule is heterologous to the sample.
  • RNA-comprising template molecule comprises (i) a 3' portion complementary to the short RNA target and composed of RNA and (ii) a 5' portion that forms a 5' overhang when the RNA-comprising template molecule is annealed to the short RNA target molecule.
  • the 5' portion is DNA except for a 1-2 ribonucleotide portion of the 5' portion linked to the '3 portion.
  • the first primer is the 5' DNA portion that is released by the RNaseH activity.
  • the contacting of the sample comprises contacting the sample with the short RNA target and a plurality of RNA-comprising template molecules having a 5' portion and a 3' portion, wherein said 3' portion comprises a degenerate sequence of at least 4 nucleotides such that the plurality comprises a variety of RNA-comprising molecules having different 3 portions and identical 5 portions.
  • a RNaseH enzyme is from the reverse transcriptase.
  • the amplifying comprises generating an amplicon
  • the method further comprises nucleotide sequencing the amplicon.
  • the method as defined above further comprising quantifying the amount of short RNA target molecule in the sample.
  • a method of amplifying a non-polyA tailed RNA less than 30 nucleotides long comprising reverse transcribing the RNA by extending a first primer having at least 6 contiguous nucleotides complementary to the RNA, to produce a first strand cDNA comprising a sequence at least 10 nucleotides long, in addition to the primer sequence, that is complementary to the RNA , amplifying the first strand cDNA with a DNA polymerase-DNA binding domain fusion to extend a second primer, wherein the DNA polymerase has a foot print of 6-10 base pairs and the DNA binding domain has a footprint of 3-5 base pairs and wherein the second primer comprises contiguous base pairs complementary to a 3' region of the first strand cDNA to produce a double-stranded cDNA.
  • the first primer comprises a 5' portion not complementary to the RNA.
  • the second primer comprises 12-16 contiguous base pairs complementary to a 3' region of the first strand cDNA
  • polymerase is a Taq Stoffel fragment or an analog thereof.
  • DNA binding domain is an Sso7d DNA binding domain.
  • RNA is a miRNA, snoRNA, piRNA, or lncRNA.
  • first primer has 6-12 contiguous nucleotides complementary to the RNA.
  • the amplifying comprises generating an amplicon
  • the method further comprises nucleotide sequencing the amplicon.
  • the method as defined above further comprising quantifying the amount of the non-polyA tailed RNA in the sample.
  • a method of generating cDNA from non-polyA tailed RNA comprising adding random poly-W (A/T) or poly-S (G/C) nucleotide sequences to the 3' end of target non-polyA tailed RNA with a terminal transferase to form RNA molecules comprising degenerate nucleotide sequences at the 3' end of the molecules; submitting the RNA molecules comprising the random poly-W or poly-S nucleotide sequences to conditions such that the RNA molecules comprising the random poly-W or poly-S nucleotide sequences anneal to form a duplex of a first and second RNA molecule annealed to each other via the random poly-W or poly-S nucleotide sequences; and extending the 3' ends of the molecules in the duplex with a DNA polymerase to form (i) a first cDNA complementary to the first RNA molecule of the duplex and (ii) a second cDNA complementary to the second
  • RNA is a miRNA, snoRNA, piRNA, or lncRNA.
  • the method as defined above further comprising amplifying the first or second cDNA with a primer that anneals to the complement of the degenerate sequence.
  • adding further comprises contacting the non-polyA tailed RNA with a poly A polymerase.
  • the amplifying comprises generating an amplicon
  • the method further comprises nucleotide sequencing the amplicon.

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CN109234813B (zh) * 2018-09-11 2021-11-16 南京迪康金诺生物技术有限公司 一种构建链特异rna文库的方法及应用
CN110055316A (zh) * 2019-03-28 2019-07-26 天津大学 一种用于检测微小rna的由末端脱氧核苷酸转移酶介导的反转录pcr方法
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